Characterisation of phenolic extracts from olive pulp and ... - ESAC

Journal of the Science of Food and Agriculture J Sci Food Agric 85:21–32 (2005)
DOI: 10.1002/jsfa.1925
Characterisation of phenolic extracts from
olive pulp and olive pomace by electrospray
mass spectrometry
Susana M Cardoso, 1,2 Sylvain Guyot, 3 Nathalie Marnet, 3 José A Lopes-da-Silva, 1
Catherine MGC Renard 3 and Manuel A Coimbra 1∗
1 Departamento de Química, Universidade de Aveiro, P-3810-193 Aveiro, Portugal
2 Escola Superior Agrária de Bragança, Instituto Superior Politécnico de Bragança, P-5301-855 Bragança, Portugal
3 Unité de Recherches Cidricoles—Biotransformations des Fruits et Légumes, INRA, BP 35327, F-35653 Le Rheu Cédex, France
Abstract: Methanol extracts of olive pomace (two-phase olive oil extraction) and olive pulp were analysed
by reverse phase HPLC and the eluted fractions were characterised by electrospray ionisation mass
spectrometry. This technique allowed the identification of some common phenolic compounds, namely,
verbascoside, rutin, caffeoyl-quinic acid, luteolin-4-glucoside and 11-methyl-oleoside. Hydroxytyrosol-1 ′ -
β-glucoside, luteolin-7-rutinoside and oleoside were also detected. Moreover, this technique enabled the
identification, for the first time in Olea europaea tissues, of two oleoside derivatives, 6 ′ -β-glucopyranosyloleoside
and 6 ′ -β-rhamnopyranosyl-oleoside, and of 10-hydroxy-oleuropein. Also, an oleuropein glucoside
that had previously been identified in olive leaves was now detected in olive fruit, both in olive pulp and
olive pomace. With the exception of oleoside and oleuropein, the majority of phenolic compounds were
found to occur in equivalent amounts in olive pulp and olive pomace. Oleoside was the main phenolic
compound in olive pulp (31.6 mg g −1 ) but was reduced to 3.6 mg g −1 in olive pomace, and oleuropein
(2.7 mg g −1 in the pulp) almost disappeared (

SM Cardoso et al
biological properties such as antimicrobial, hypoglycaemic,
hypolipidemic, hypocholesterolic, antioxidant
and free radical-scavenging actions. 11 – 15 The association
of these properties with the prevention of several
diseases such as atherosclerosis and heart diseases has
raised interest in these phenolic compounds.
The analysis of phenolic extracts from olive and
related products has been mostly performed by
reverse phase HPLC coupled with diode array
detection (DAD). More recently, the association of
this methodology with ionisation mass spectrometry
has proved to be useful in the identification of the
16 – 20
major compounds in olive phenolic extracts.
Moreover, this methodology has been of great help
in the identification of new compounds even when
present in trace amounts. 21 In order to contribute to
the knowledge of the phenolic compounds present
in olive pomace, the reversed phase HPLC/DAD
separation of a methanol extract was performed, the
collected fractions were characterised by electrospray
ionisation mass spectrometry (ESI-MS n ) and the
structures of new compounds were identified by MS 2
and MS 3 experiments.
MATERIALS AND METHODS
Phenolic standards
Caffeoyl-quinic acid, vanillic acid, protocatechuic
acid, syringic acid, oleuropein, luteolin, luteolin-7-
glucoside, cinnamic acid and tyrosol were purchased
from Sigma Chemical Co (St Louis, MO, USA).
Solvents and reagents
n-Hexane, methanol, acetone and acetonitrile, all of
chromatographic grade, were purchased from Biosolve
BV (Valkenswaard, The Netherlands). Glacial
acetic acid was also purchased from Biosolve Ltd.
Folin–Ciocalteu reagent was purchased from Merck
(Darmstadt, Germany). Deionised water was obtained
with a Milli-Q water system (Millipore, Bedford, MA,
USA).
Sample origin
The analyses were performed on two samples of
the same batch: olive pomace and olive pulp (Olea
europaea L var Verdial). The samples were collected at
Prolagar, an olive oil factory in Mirandela, Portugal. A
representative sample (2 kg, 912 olives) was collected
before processing. These olives were stoned, immersed
in 0.5% NaF solution and homogenised in a mixer. In
the factory, olives from the same batch were crushed
with a hammer mill, malaxed (slowly mixed) in a
sequential extractor at a temperature close to 40 ◦ C
for 45 min and separated from the olive oil in a
continuous two-phase centrifugation system. At this
stage the olive pomace was collected. After collection,
this residue was immediately immersed in 0.5% NaF.
After arriving at the laboratory, the samples were
immediately frozen, freeze-dried and kept at −20 ◦ C
until used.
Extraction of phenolic compounds
The extraction procedure used for the olive pulp and
olive pomace was adapted from that of Guyot et al. 22
Just before extraction the freeze-dried materials were
sieved through a 700 µm filter to remove the nonpulverised
peel and the stones that were present
in the olive pomace. Each sample (30 g of powder)
was defatted with n-hexane, which was subsequently
discarded, and the residue was extracted with 300 ml
of methanol. The solution was filtered, concentrated,
frozen at −20 ◦ C and freeze-dried to give the nonpurified
methanol extract. The resulting residue was
extracted with acetone/water (6:4 v/v). Acetone was
eliminated as described for methanol and the aqueous
solution was frozen and freeze-dried to obtain the
non-purified aqueous acetone extract. The insoluble
residue was abundantly washed with water, frozen and
freeze-dried.
Estimation of amount of smashed seed hull in
olive pomace
To relate the amount of material extracted from
olive pomace with olive pulp, a procedure to allow
the estimation of the amount of olive hull in olive
pomace material was designed. As this lignified
material contains low amounts of the typical phenolic
compounds found in the pulp, 5,23 it should be omitted
from the basis of calculation of starting material rich in
phenolic compounds. An olive pomace representative
sample was freeze-dried and defatted and the resulting
residue was sieved through a 300 µm filter in order
to separate the smashed seed and stone particles
still present from the pulp material. The seeds and
stones that are resistant to the grinding and malaxing,
remain as coarser particles which are retained on the
sieve, while the purified pulp passes through. The
sieving through the 300 µm filter was only possible
after defatting, since otherwise the fat present in the
material would immediately have collapsed the fine
pores of the filter. This procedure resulted in a residue
without visible smashed particles, which accounted for
90.1% of the defatted residue.
Purification of methanol and acetone extracts
The purification step was performed on Sep Pack
C18 cartridges (5 g, Waters, Milford, MA, USA).
The cartridges were preconditioned by sequential
treatment with methanol, H 2 O and 2% acetic
acid. Two fractions of the phenolic compounds
were recovered by elution of the cartridges with
methanol/water/acetic acid (50:48:2 v/v/v) followed
by methanol/acetic acid (98:2 v/v). The fractions
corresponding to 50 and 100% methanol extractions
(MeOH 50 and MeOH 100 respectively) were
concentrated to an aqueous suspension, frozen and
freeze-dried.
Colorimetric quantification of total phenolic
compounds by Folin–Ciocalteu method
The total concentration of phenolic compounds in the
non-purified and purified extracts was determined by
22 J Sci Food Agric 85:21–32 (2005)

Phenolic compounds of olive pomace
an adaptation of the Folin–Ciocalteu method 24 by
dispersing the non-purified and purified extracts by
sonication in aqueous acetic acid (2.5% v/v) and using
a calibration curve of oleuropein standard (0–70 µg).
Reverse phase HPLC conditions
HPLC analysis was performed using a Waters 2690
separation module equipped with an autosampler
and a cooling system, set to 4 ◦ C, and a Waters
996 photodiode array detector. Data acquisition and
remote control of the HPLC system were done
by Millennium 32 version 3.20 software (Waters,
Milford, MA, USA). The column was a 250 mm ×
4mm id, 5µm bead diameter, end-capped Purospher
RP 18 column (Merck) maintained at 30 ◦ C. The
mobile phase comprised (A) 2.5% acetic acid and
(B) acetonitrile, which were previously degassed and
then continuously sparged with high-purity helium
during analysis to prevent air resaturation. The solvent
gradient started with 97% A and 3% B, reaching 91%
A at 4 min, 85% A at 15 min, 79% A at 75 min,
70% A at 80 min and 10% A at 85 min, followed by
an isocratic plateau for 5 min and a return to initial
conditions.
For the HPLC analysis the purified methanol
extracts (5 mg) were dissolved in 1 ml of methanol/-
acetic acid (99:1 v/v). All samples were filtered through
a0.45 µm Teflon membrane (Millipore) and 10 µl of
each solution was injected.
HPLC characterisation of phenolic compounds
Compounds for which standards were available were
first identified by comparison of the retention times
and UV/vis spectra of the corresponding peaks. As
on-line LC/MS does not give enough time to examine
in detail the MS n patterns of the various fragments,
the 27 peak-forming fractions were collected prior to
their characterisation by electrospray ionisation mass
spectrometry (ESI-MS n ).
HPLC quantification of phenolic compounds
Quantification of the identified compounds was performed
by correlating the measured peak area with
the calibration curves obtained with reference compounds.
Oleuropein and hydroxytyrosol glucoside
were quantified according to their absorbances at
280 nm. In accordance with Mulinacci et al, 8 hydroxytyrosol
glucoside was quantified using tyrosol as
reference compound. Oleoside and its derivatives
were evaluated at 240 nm using oleuropein as reference.
Caffeoyl-quinic acid was evaluated at 320 nm
using caffeoyl-quinic acid as reference. The flavones
luteolin-7-glucoside, luteolin-4-glucoside, luteolin-7-
rutinoside and rutin were evaluated at 340 nm and
expressed with the extinction coefficient of luteolin-7-
glucoside.
ESI-MS
The mass spectrometry system was an LCQ DECA
ion trap mass spectrometer (Thermofinnigan, San
Jose, CA, USA) equipped with an ESI source and
run by Xcalibur ® (Thermofinnigan, San Jose, CA,
USA) version 1.2 software. Infusion analyses were
performed in negative mode with an ion spray voltage
of approximately 4500 kV, a −60 V orifice voltage, a
225 ◦ C capillary temperature, a 50 au (arbitrary units)
sheath nitrogen gas flow rate and a nominal mass
range up to m/z 1800. Although phenolic compounds
give lower-intensity peaks in negative than in positive
mode, negative ion electrospray was used because
cleaner spectra were obtained. Samples corresponding
to collected HPLC peaks were directly introduced into
the ESI source by a built-in syringe pump at a flow
rate of 10 µl min −1 .
RESULTS AND DISCUSSION
Isolation and purification of phenolic compounds
The yields of mass and phenolic compounds extracted
from olive pulp and olive pomace using methanol and
aqueous acetone are presented in Table 1. Ponderal
yields were calculated on a dried, defatted and
dehulled basis to facilitate comparisons between olive
pulp and olive pomace. Indeed, the two samples had
different oil contents and, in contrast with the olive
pulp, the olive pomace still contained smashed seed
hulls, the weight of which was estimated as 9.9% of
defatted olive pomace.
For both samples the methanol extract was the
most significant one, representing 68 and 66% of
the dried, defatted and dehulled olive pulp and olive
pomace respectively. The aqueous acetone extract of
both samples consisted of about 10% of the methanol
extract. The remaining residues represented 23 and
28% of the olive pulp and olive pomace respectively.
Mass recovery of the extracts and residues was about
98% for olive pulp and total for olive pomace.
The total phenolics of each extract were expressed
as oleuropein equivalents and the values are shown in
Table 1. Depending on the considered material (pulp
or pomace), the non-purified methanol and aqueous
acetone extracts showed total polyphenol proportions
in the range of 20–36%. After C18 cartridge
purification the phenolic content was raised, with a
phenolic recovery of 97–99% in the methanol extracts
(MeOH 50 and MeOH 100) and total recovery in
the acetone extracts (Acetone 50 and Acetone 100).
The sum of the amounts of phenolic compounds
obtained in methanol and acetone extracts, calculated
on a dried, defatted and dehulled starting material
basis, was similar (154 mg g −1 for olive pulp and
146 mg g −1 for olive pomace). For both samples,
most of the phenolic material was present in the
MeOH 50 fraction, which represented 73% of the
extractable phenolics and was therefore chosen for
further investigation.
Separation of phenolic compounds by reverse
phase HPLC
The MeOH 50 fractions of olive pomace and olive
pulp were fractionated and analysed by HPLC/DAD
J Sci Food Agric 85:21–32 (2005) 23

SM Cardoso et al
Table 1. Yields of mass and phenolic compounds in extracts and purified fractions (C18 cartridges)
Fraction
Mass (% of dry weight)
Total phenolics a
(mg g −1 fraction)
Total phenolics
recovered
(%)
Total phenolics b
(mg g −1 pulp
or pomace)
Olive pulp
Non-purified methanol extract 68 c 204 — —
MeOH 50 22 d 752 82 111
MeOH 100 5 d 716 17 23
Non-purified acetone extract 7 c 295 — —
Acetone 50 33 d 726 81 16
Acetone 100 13 d 459 20 4
Olive pomace
Non-purified methanol extract 66 c 198 — —
MeOH 50 18 d 904 83 106
MeOH 100 5 d 601 14 18
Non-purified acetone extract 6 c 356 — —
Acetone 50 35 d 830 82 18
Acetone 100 14 d 480 19 4
a Values expressed as oleuropein equivalents, as result of Folin–Ciocalteu assay.
b Values expressed as mg phenolic compounds (oleuropein equivalents, as determined by Folin-Ciocalteu assay) g −1 dried, defatted and dehulled
material.
c Yield expressed as percentage of dried, defatted and dehulled starting material (olive pulp or olive pomace).
d Yield expressed as percentage of non-purified extract (methanol or acetone).
100
1
8
10
18
olive pulp
Relative absorbance (%)
75
50
25
2,3
6,7
4 5
9
12,13
17
11 14 16
15
19
20
22
21
23 25
24 26
0
100
olive pomace
Relative absorbance (%)
75
50
25
0
5 15 25 35 45 55 65
Time (min)
Figure 1. Chromatographic profiles of MeOH 50 fractions of olive pomace and olive pulp at 240 nm (bold curves) and 280 nm (light curves). The
numbers on the figure correspond to the fractions that were collected and analysed by ESI-MS.
and the respective chromatograms obtained at 240
and 280 nm are presented in Fig 1. To improve the
resolution of the chromatogram, fraction 27 was not
included. Both samples had a high number of resolved
fractions, suggesting a large variety of compounds.
Although reasonable chromatographic separation was
achieved, a significant rise of the baseline was observed
in the first part of the chromatogram, suggesting
co-elution of some compounds. The chromatographic
profiles of olive pomace and olive pulp at 240
and 280 nm (Fig 1) differed mostly in the relative
abundance of the various peak-forming compounds.
This was more evident at 240 nm, related to the
presence of secoiridoid derivatives, namely fractions
24 J Sci Food Agric 85:21–32 (2005)

Phenolic compounds of olive pomace
Table 2. Identification of HPLC-eluting fractions from MeOH 50 (pulp or pomace) extract and correspondence with results obtained by mass
spectrometry analysis
Fraction
number
Retention
Predominant
(min) Ident a [M − H] − by ESI-MS b
time
negative ion
Main fragments
Compound
1 10.0 B 315 153, 135, 179, 161 Hydroxytyrosol-1 ′ -β-Glucoside
2a 11.2 — 421 389, 241, 239, 165, 195 Unknown
2b 11.2 — 407 389, 375, 357, 313, 161 Unknown
3 11.7 — NI —
4 12.8 — NI —
5 14.5 — 763 565, 341 Unknown
6 15.5 C 353 191, 179, 161 Caffeoyl-quinic acid
7 15.8 — NI —
8 16.2 B, C 389 345, 209 Oleoside
9 17.2 — NI —
10 17.8 B 595 Unknown
11 20.7 — 377 197, 179, 153 Oleuropein aglycone derivative
12 22.9 — 383; 257 Unknown
13a 23.3 B 403 11-Methyl-oleoside
13b 23.3 B 151 123, 108 4-Hydroxyphenylacetic acid
14 24.7 — NI —
15a 28.5 C 555 537, 403, 393 Unknown c
15b 28.5 — 579 337, 547, 561, 529 Unknown
16 30.6 B 609 301, 179 Rutin
17 31.0 B 593 447, 285 Luteolin-7-rutinoside
18a 32.6 A 447 285 Luteolin-7-glucoside
18b 32.6 B 623 461, 315, 135, 161, 297 Verbascoside
19 33.3 B 593 447, 285 Luteolin-7-rutinoside (isomer)
20 35.3 B, C 701 539, 377, 307 Oleuropein glucoside
21 43.2 B 447 285 Luteolin-4-glucoside
22 44.6 C 551 507, 341, 532, 389, 281 Unknown c
23 47.2 — 335 199, 181, 153 Unknown
24 48.9 — 663 479, 295 Unknown
25 53.9 A 539 377, 197, 153 Oleuropein
26 59.7 C 535 491, 389, 265, 325 Unknown c
27 75.2 A 285 Luteolin
a Identification was based on the following: A, retention time and DAD spectrum consistent with those of authentic standard; B, MS data consistent
with literature; C, MS with fragmentation.
b Ordered by decreasing intensity.
c Compounds to be elucidated in the present study.
NI, no main [M − H] − identified.
The notation a,b for a peak number in the fraction number column indicates co-eluted compounds.
8 and 10 (240 nm) and fraction 25 (240 and 280 nm),
which were detected as intense peaks only in olive
pulp.
Only three of the 27 fractions matched with the
nine standard compounds used (see Table 2), namely
fractions 18, 25 and 27, attributed to luteolin-
7-glucoside, oleuropein and luteolin respectively,
which correspond to compounds usually found in
olives. 23,25,26 However, tyrosol, vanillic acid and
syringic acid, which were among the standards and
are also frequently found in phenolic extracts from
olive, 17,27 although not always, 4,6 were not detected.
The HPLC-eluting fractions were analysed by ESI-
MS to complete the identification of the phenolic
compounds in olive pulp and olive pomace.
Analysis of HPLC fractions by ESI-MS
The identification of the corresponding compounds
was based on the search of the main [M − H] − ion
together with the interpretation of its collision-induced
dissociation (CID) fragments. Table 2 summarises the
data obtained for each of the analysed fractions. In
some fractions the ionic species [M − H] − was not
observed (marked with ‘NI’ in Table 2), probably
because the solvent and/or MS conditions were not
favourable to its ionisation. Also, 11 of the 26 detected
molecular ions were compounds not yet known in O
europaea.
The compounds previously identified by HPLC/-
DAD were confirmed by ESI-MS. Luteolin (peak 27)
showed an intense molecular ion at m/z 285 and for its
derivative luteolin-7-glucoside (peak 18a) a molecular
ion at m/z 447 and also a strong fragment at m/z
285 were obtained. The identification of peak 25
as oleuropein was corroborated by detection of the
molecular ion at m/z 539 and its aglycone fragment
at m/z 377. Fraction 11 shows ions characteristic of
oleuropein aglycone or one of its isoforms. 28
J Sci Food Agric 85:21–32 (2005) 25

SM Cardoso et al
The comparison of the ESI-MS data with
literature data also allowed the identification of
hydroxytyrosol-1 ′ -β-glucoside (peak 1), 11-methyloleoside
(peak 13a), hydroxylphenylacetic acid (peak
13b), three derivatives of luteolin (peaks 17, 19 and
21), verbascoside (peak 18b), oleoside (fraction 8) and
oleuropein glucoside (fraction 20).
The main compound in fraction 1 had a molecular
ion at m/z 315 and fragment ions at m/z 153,
135, 180 and 161, which suggested the presence
of a hydroxytyrosol hexoside. To our knowledge,
three isomers of hydroxytyrosol glucoside have been
characterised by NMR in olive fruit and olive oil. 29
These isomers have also been detected in different
table olive varieties, 30 in which hydroxytyrosol-4-βglucoside
was the most abundant compound. This
isomer was also the only one detected by Romero et al 9
in olive pulp, olive pomace and waste waters. However,
the similarity between the fragmentation profile of the
molecular ion at m/z 315 in fraction 1 to that published
by De Nino et al 21 allowed us to infer that this
compound should be hydroxytyrosol-1 ′ -β-glucoside.
Fractions 13a and 13b were attributed to 11-methyloleoside
and 4-hydroxylphenylacetic acid respectively
based on their specific and characteristic molecular
ions described in the literature for Oeuropaea. 23 The
high absorbance at 240 nm of fraction 10 can possibly
be attributed to a secoiridoid derivative.
The presence of a fragment at m/z 285 is
diagnostic of luteolin derivatives. According to the
molecular ion at m/z 593 and its main fragments
observed for both fractions 17 and 19, luteolin-
7-rutinoside could be proposed for both fractions.
To our knowledge, the flavone luteolin-7-rutinoside
was previously detected only in olive leaves 23 and
its ESI-MS data were similar to those of peaks
17 and 19. Since the luteolin-7-rutinoside detected
by Ryan et al 23 eluted before luteolin-7-glucoside in
HPLC reverse phase conditions, the same compound
was tentatively attributed to fraction 17. Thus
fraction 19 may correspond to a non-described
isomer of that compound, probably with a different
linkage position to the sugar. The MS analysis
of fraction 21 demonstrated a molecular ion at
m/z 447, suggesting the presence of a luteolin
hexoside. Four luteolin glucosides have already
been detected in olives: luteolin-7-glucoside and
its three isomers 23 luteolin-4-glucoside, luteolin-6-
glucoside and luteolin-8-glucoside. According to those
authors, luteolin-4-glucoside was the only one eluting
after luteolin-7-glucoside in HPLC reverse phase
conditions and that was the reason why fraction 21
was attributed to that isomer.
ESI-MS of fraction 18b indicated a molecular ion at
m/z 623 and various fragments that are in accordance
with the fragmentation of verbascoside. These results
were also corroborated with the fragmentation profile
of verbascoside described by Ryan et al. 18
The comparison of the MS data with literature
data was also possible for the compounds detected
in fraction 8 (oleoside) and fraction 20 (oleuropein
glucoside). However, as they had not been detected
previously in olive pulp, the interpretation of their
structures will be discussed in more detail. Also, the
structures of some new oleoside derivatives corresponding
to fractions 15a, 22 and 26 will be elucidated.
Structure determination of fraction 8
The mass spectrum of fraction 8, eluted at 16.2 min,
displayed an intense peak at m/z 389 which formed two
major fragments by CID, one at m/z 345 and the other
at m/z 209 (Fig 2). The former corresponded to the
loss of 44 Da, which can be justified by the elimination
of a CO 2 molecule of a carboxylic group, and the latter
can be attributed to the Z fragment of a hexose (loss of
180 Da). This hexose residue was attributed to glucose
in accordance with the literature. 16,23 The presence of
a hexose moiety was also supported by the detection of
minor ionic species at m/z 161 and 179 in the ESI-MS 2
spectrum shown in Fig 2 (inset) and major ones in the
ESI-MS 3 spectrum of the ion at m/z 345 (results
not shown). These results are in agreement with the
100
389
100
345
Intensity (%)
75
50
25
Intensity (%)
75
50
209
25
389
161
179
0
150 250 350 450
m/z
0
150 500 850 1200
m/z
Figure 2. ESI-MS spectrum of fraction 8 and (inset) ESI-MS 2 spectrum of molecular ion at m/z 389.
26 J Sci Food Agric 85:21–32 (2005)

Phenolic compounds of olive pomace
presence of oleoside, which has a molecular mass of
390 Da (represented by the fragment at m/z 389 in
Fig 4). This compound has previously been detected
in olive leaves by the use of ESI-MS in positive mode. 21
However, to our knowledge, its presence in olive fruit
is now demonstrated for the first time.
Structure determination of fraction 22
A derivative of oleoside, also not yet reported to occur
in Oeuropaea, was found in fraction 22. The mass
spectrum of that fraction showed a strong peak at
m/z 551 (Fig 3a) whose MS 2 fragmentation spectrum
indicated various ionic species (Fig 3a, inset). As
discussed for oleoside, the principal fragment was
originated by the loss of 44 Da, giving rise to the
intense signal at m/z 507. The ionic species at m/z 389,
representing the oleoside structure, was also observed
in the ESI-MS spectrum of the molecular ion. Its
formation was accomplished by the loss of a hexose
moiety (162 Da), suggesting that the compound was
a hexoside derivative of oleoside. Moreover, the
presence of a fragment at m/z 341, which corresponds
to a disaccharide, indicated that this hexose molecule
should be linked to the sugar moiety of oleoside
(tentative structure of the molecular ion in Fig 4).
The presence of the fragment at m/z 251 is
31 – 33
characteristic of a (1→6) disaccharide, and a
low-intensity signal at m/z 221 can be diagnostic of a β
isomer. 33 According to Fig 3b, it is probable that the
oleoside derivative detected in fraction 22 was a 6 ′ -βhexopyranosyl-oleoside,
possibly 6 ′ -β-glucopyranosyloleoside,
with a scheme of fragmentation in negative
mode as represented in Fig 4.
Structure determination of fraction 26
Fraction 26 of the chromatogram was a distinct and
relatively intense peak. Its mass spectrum showed
a high-intensity ion at m/z 535 that has not been
detected so far in Oeuropaea(Fig 5). The ESI-MS 2
spectrum of that ion (Fig 5, inset) demonstrated
some similarities to the spectral profile of the two
compounds already described (Figs 2 and 3a, insets).
Namely, the main signal was obtained by the loss
of 44 Da (ion at m/z 491), and an ionic species
corresponding to the oleoside ion (m/z 389) was
also detected. In this case the oleoside fragment
was originated by the elimination of 146 Da, which
can be justified by the Y fragmentation of a
deoxyhexose molecule (fragment Y in the tentative
structure represented in Fig 5). In agreement with
this hypothesis, in Fig 5 (inset) the signal observed
Intensity (%)
100
75
50
25
551
Intensity (%)
100
75
50
25
507
341
533
389
551
251 281
0
150 300 450 600
m/z
a
0
300 600 900 1200
m/z
100
75
161
345
341
Intensity (%)
b
50
281
251
323
393
25
221
489507
179
463
0
150 250 350 450 550
m/z
Figure 3. Mass spectra of fraction 22: (a) ESI-MS spectrum and (inset) [551] ESI-MS 2 molecular ion spectrum; (b) [551→507] ESI-MS 3 spectrum.
J Sci Food Agric 85:21–32 (2005) 27

SM Cardoso et al
HO
HO
m/z 341
OH
O
OH
HO
HO
O
HO
HO
Z
O
Z
OH
HO
HO
OH
O
OH
HO
HO
Y
O
O
OH
O
O
(-H) -
m/z 551 O
Y
(-H) - OH
OH
O
(-H) -
HO
O
OH
- CO 2 HO
OH
O
O OH
OH
m/z 389
O
O
O
OH
O
(-H) -
HO
O
HO
OH
O OH
m/z 507
Z
O
OH
O
OH
Y
HO
HO
OH
O
O
OH
O
m/z 345
OH
O
(-H) -
Figure 4. Proposed scheme for fragmentation of molecular ion at m/z 551 of fraction 22.
100
491
75
Intensity (%)
100
75
50
25
535
HO
HO
Intensity (%)
OH
O
HO
HO
50
25
389
265 325
235 345
535
0
150 300 450 600
m/z
(m/z = 389)
Y
O
O
HO
(m/z = 325)
Z
O
O
O
OH
O
(m/z = 491)
OH
0
300 600 900 1200
m/z
Figure 5. ESI-MS spectrum of fraction 26 and (inset) ESI-MS 2 spectrum of molecular ion at m/z 491. The tentative structure of the compound is
also shown.
at m/z 325 (fragment Z) can correspond to a
deoxyhexose—hexose disaccharide residue, which
should correspond to a rhamnose-glucose residue,
one of the most common disaccharides found in
phenolic compounds. 34 The 535 MS 2 spectrum also
showed the fragments of the disaccharide moiety that
correspond to a pattern similar to that for glucose-
(1→6)-glucose disaccharide residue, suggesting the
presence of a rhamnose-(1→6)-glucose. In this
manner, it can be concluded that this new compound
is a 6 ′ -deoxyhexopyranosyl-oleoside, possibly 6 ′ -βrhamnopyranosyl-oleoside.
Structure determination of fraction 20
The ESI-MS analysis of fraction 20 showed an
[M − H] − ion at m/z 701 (results not shown). Its MS 2
28 J Sci Food Agric 85:21–32 (2005)

Phenolic compounds of olive pomace
a
100
539
75
Intensity (%)
50
377
701
25
307 162 Da 162 Da
0
300 400 500 600 700 800
m/z
b
HO
HO
OH
O
OH
Y
O
O
O
O
O
O
Isomer I
Y
HO
O
OH
O
OH
OH
OH
HO
HO
OH
O
OH
Y 1
O
HO
OH Y 2
O
O
OH
O
Isomer II
O
O
O
O
OH
OH
Figure 6. (a) ESI-MS 2 spectrum of molecular ion at m/z 701. (b) Structure of two diglucoside homologues of oleuropein.
fragmentation profile showed a main ionic species at
m/z 539 that was formed by the loss of 162 Da, and
another intense peak at m/z 377 indicative of the
elimination of another hexose unit (Fig 6a). These
two main fragments correspond to oleuropein and its
aglycone respectively and together they support the
hypothesis of a hexose derivative of the oleuropein
structure. To our knowledge, hexose derivatives of
oleuropein have never been described in olive fruit.
However, angustifolioside A (isomer I represented in
Fig 6b) was already described to occur in the family
Oleaceae. 35 Also, De Nino et al 16 have proposed
the presence of the same isomer in olive leaves,
although their results did not allow the exact structural
determination, and the exclusion of isomer II. In the
present study the loss of 162 Da of the molecular
ion at m/z 701 can fit for both isomer structures:
the consecutive or simultaneous elimination of a Y-
type hexose fragment would be possible for the two
compounds, explaining the fragments at m/z 539 and
377. However, from the oleoside derivatives discussed
in Figs 3 and 5, it can be expected that, if isomer II was
present, the O-dihexosyl ion (at m/z 341) together with
its fragments would have appeared in the ESI-MS 2
spectrum of the molecular ion. Yet, the total absence
of those species was confirmed, indicating that the
isomer present in fraction 20 is angustifolioside A
(isomer I in Fig 6b). These results together with those
of De Nino et al 16 suggest that Oeuropaeahas the
same glucoside derivative of oleuropein that is present
in Fraxinus angustifolia. 35
Structure determination of fraction 15a
The MS analysis of fraction 15a showed a predominant
[M − H] − signal at m/z 555. As for the oleoside
derivatives, it was not possible to find any MS data in
the literature about this compound. Alternatively, its
structure elucidation was only based on its ESI-MS n
analysis. Fig 7 shows the ESI-MS 2 spectrum of the
ion at m/z 555. The main fragment ion represented in
the spectrum was obtained by the loss of 18 Da (ion
at m/z 537), suggesting that the compound has an OH
group that is easily removed. Also, another two intense
peaks at m/z 393 and 403 could be observed. The first
corresponded to the aglycone (loss of 162 Da) and
the latter was equivalent to the mass of an 11-methyloleoside
moiety. The aglycone at m/z 393 was already
detected as 10-hydroxy-oleuropein aglycone in olive
oil using mass spectrometry, 36 which indicates that
this compound should be 10-hydroxy-oleuropein. In
this manner the fragments at m/z 537 and 393 arose
from the loss of water from the 10-OH group and
from the Y-type cleavage of the molecule respectively.
The fragment ion at m/z 403 could be originated by
a cleavage X together with the loss of water. To our
knowledge, 10-hydroxy-oleuropein has not previously
been detected in any tissue of Oeuropaea.
J Sci Food Agric 85:21–32 (2005) 29

SM Cardoso et al
Intensity (%)
100
75
50
HO
HO
(m/z = 393)
OH Y
O
O
OH
O
OH
X
O
O
O
O
403
393
OH
OH
537
555
25
179
161
291 375
0
150 300 450 600
m/z
512
523
Figure 7. ESI-MS 2 spectrum of molecular ion at m/z 555. The structure of the molecular compound is also represented.
Table 3. Quantification of main identified compounds (mg g −1 ) in MeOH 50 extracts of olive pulp and olive pomace
Fraction
number Compound Olive pulp Olive pomace
1 Hydroxytyrosol-1 ′ -β-glucoside 6.4 (0.4) 6.5 (0.3)
6 Caffeoyl-quinic acid 0.10 (0.01) 0.09 (0.01)
8 Oleoside 31.6 (7.9) 3.6 (0.1)
16 Rutin 0.73 (0.03) 0.66 (0.03)
17 Luteolin-7-rutinoside 0.10 (0.01) 0.32 (0.01)
18 a Luteolin-7-glucoside + verbascoside 2.0 (0.1) 2.1 (0.0)
19 Luteolin-7-rutinoside (isomer) 0.41 (0.03) 0.44 (0.01)
21 Luteolin-4-glucoside 0.37 (0.03) 0.47 (0.01)
22 6 ′ -β-Glucopyranosyl-oleoside 6.6 (0.2) 5.0 (0.0)
25 Oleuropein 2.7 (0.14) V
26 6 ′ -β-Rhamnopyranosyl-oleoside 3.8 (0.2) 6.5 (0.1)
Total 54.8 25.7
Phenolic compounds were determined as the mean value of two independent assays measured in duplicate. Values in parentheses represent the
standard deviation. Values are expressed as mg phenolic g −1 dried, defatted and dehulled starting material. V, vestigial quantity.
a Quantified as luteolin-7-glucoside.
By the same reasoning (loss of respectively 32, ie
a methoxyl, and 18, ie a hydroxyl, to give the ion at
389), fractions 2a and 2b were respectively hydroxy
and methoxy derivatives of oleoside, which were not
investigated further.
Quantification of main identified compounds
separated by HPLC
The amounts of the major identified compounds
are shown in Table 3. Demethyloleuropein, tyrosol,
hydroxytyrosol and vanillic acid, which are frequently
detected in olive pulp, were not found in this sample.
As the profile of olive pulp phenolics and derivatives
can be influenced by various factors such as olive
cultivar, climatic conditions, degree of maturation and
agronomic practices, 9 the absence of these compounds
can be accepted as possible. According to Table 1, the
compounds identified in Table 3 represent 49% of the
total phenolics present in the MeOH 50 extract of olive
pulp, but only 24% of those present in the MeOH 50
extract of olive pomace. These results can be related
to the possible modification of olive phenolics during
olive oil extraction. The amounts of hydroxytyrosol-
1 ′ -β-glucoside, caffeoyl-quinic acid and flavones, with
the exception of rutin, were not greatly affected by the
olive oil extraction process (Table 3). The secoiridoids
were more affected by the extraction process: oleuropein
was one of the main compounds in olive pulp
(2.7mgg −1 sample) but only a vestigial compound
in olive pomace. This result suggests that oleuropein
could be extracted to the oil phase, which is supported
by its detection in the oil, 37 although this
compound is mostly soluble in water. Alternatively,
oleuropein could be degraded during the crushing
and malaxation of the olives, as its glycosydic linkage
is easily hydrolysed by β-glucosidases, producing
oleuropein aglycone which is more hydrophobic and
consequently more soluble in the oil. This is corroborated
by the frequent detection of oleuropein aglycone
and its isomer 3,4-(dihydroxyphenyl)ethanol elenoic
acid ester (3,4-DHPEA-EA) in olive oil. 9–11,26,38,39
Moreover, it is well known that oleuropein aglycone
can be modified due to the keto–enol tautomeric equilibrium
that involves the ring opening of secoiridoids,
30 J Sci Food Agric 85:21–32 (2005)

Phenolic compounds of olive pomace
usually originating secoiridoid derivatives, such as the
dialdehydic form of elenoic acid linked to hydroxytyrosol
(3,4-DHPEA-EDA) or to tyrosol (p-DHPEA-
EDA). 9–11,26,38,39 In this way the lower quantity of
oleuropein in olive pomace, when compared with olive
pulp, is probably correlated with the formation of these
compounds, which are major phenolics in olive oil.
Oleoside and its derivatives were very significant
compounds in the phenolic extract of both samples.
In olive pulp, oleoside was evaluated at 31.6mgg −1
sample. However, its concentration was drastically
diminished to 3.6mgg −1 in the olive pomace,
indicating a loss of approximately 89% during oil
extraction. As for oleuropein, this can probably be
explained by its degradation during the malaxation
of the pastes, with a concomitant loss to the oil or
accumulation of newly formed oleoside derivatives
in the olive pomace, which were not quantified or
detected at 280 nm. The accumulation of oleoside
derivatives in the olive pomace is in accordance
with the different profiles demonstrated for the
two samples at 240 nm (Fig 1). Compounds such
as 6 ′ -β-rhamnopyranosyl-oleoside and the majority
of luteolin derivatives, namely luteolin-7-rutinoside,
showed higher concentrations in olive pomace than
in olive pulp. As the contribution of the reserve
endosperm was not taken into account in the
calculations of olive pomace phenolics, the higher
amounts of these compounds suggest their presence
in this tissue.
CONCLUSION
The analysis of the methanol extract by ESI-MS n
allowed the detection of the common phenolic
compounds but also detected unusual ones. Moreover,
these techniques were very useful in the structure
elucidation of new compounds, which were mainly
hexoside derivatives of oleoside and oleuropein.
The described data surely contribute to a better
understanding of phenolic extracts from olive and its
residue obtained after olive oil extraction. Also, most
of the phenolic compounds, including hydroxytyrosol
glucoside, which can have biological activities, are not
degraded during olive oil extraction, suggesting that
the olive pomace from the two-phase system can be a
good source of those compounds, as is olive pulp.
ACKNOWLEDGEMENTS
The authors acknowledge FCT (Portugal) and the
University of Aveiro for funding Research Unit
62/94 ‘Química Orgânica, Produtos Naturais e Agro-
Alimentares’, and Fundação Calouste Gulbenkian.
Susana Cardoso was supported by a PhD grant
(PRODEP III 5.3/N/199.006/00).
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